There is extensive interest in the development of blood substitutes and blood plasma expanders which can be easily prepared and used in place of donated blood. To be effective, such a substitute should be characterized by a sufficiently high oxygen-binding capacity for use under normal environmental conditions. It also should not be subject to renal elimination.
One type of substitute that has generated significant interest is modified hemoglobins A natural mammalian hemoglobin is a tetramer, i.e. it is characterized by four polypeptide chains, two identical alpha chains and two identical beta chains, that are noncovalently linked together. In plasma, oxygenated hemoglobin has a tendency to split into dimers, each of which is small enough to be filtered by the kidneys and excreted Such dimers thus potentially cause renal damage and have significantly decreased intravascular retention time.
A modified hemoglobin that has been described in the art is alpha, alpha-cross-linked hemoglobin. Such modified hemoglobin is described in U.S. Pat. Nos. 4,598,064 and 4,600,531 issued to Walder. The two alpha chains of the tetrameric hemoglobin molecule are cross-linked, specifically at Lys 99 Alpha 1 and Lys 99 Alpha 2. The cross-linking is effected by reaction of the hemoglobin with a bis(salicyl) diester, sometimes referred to as a "diaspirin" Walder's preferred cross-linking agent is bis(3,5-dibromosalicyl) fumarate.
The preparation of aspirin-based acylating and cross-linking agents for hemoglobin is described by Zaugg et al. in "Modification of Hemoglobin with Analogs of Aspirin," J. Biol. Chem., 255:2816 (1980) and by Walder et al. in "Diaspirins that Cross-link Beta Chains of Hemoglobin: bis(3,5-dibromosalicyl) Succinate and bis(3,5-dibromosalicyl) Fumarate, Biochem., 18:4265 (1979). The procedures described in these references suffer from certain disadvantages, particularly when used for large scale syntheses. For example, both the reaction medium (benzene) and the base (N,N-dimethylaniline) recommended by Walder are recognized carcinogens. In addition, isolation of the product by precipitation with dilute aqueous hydrochloric acid gives only moderate yields (on the order of 60 to 65 percent) of a crude product which contains an unacceptably high proportion of impurities. Unfortunately, these impurities also are difficult to remove. Repeated recrystallizations give low yields (25 to 30 percent) of pure bis(3,5-dibromosalicyl) fumarate. Accordingly, improvements on the process of Walder et al. are sought.
It is an object of the present invention to provide a process for the preparation of bis(salicyl) diester cross-linking agents in which the yields of desired product are enhanced in comparison to the yields obtainable using the prior art. It is a further object of this invention to develop such a process in which the use of benzene and N,N-dimethylaniline is eliminated.